Vol. 292, No. 1, January, pp. 16-19,1992

Catalytic Properties of the Inorganic Pyrophosphatase in Rat Liver Mitochondria Elena B. Dubnova and Alexander A. IV. Belozersky Laboratory

of Molecular

A. Baykov’ Biology and Bioorganic

Chemistry, Moscow State University,

Moscow 119899, USSR

Received April 29, 1991, and in revised form August 22, 1991

Intact rat liver mitochondria have very low hydrolytic activity, if any, toward exogenous pyrophosphate. The activity can be unmasked by making mitochondria permeable to PPi by toluene treatment or disrupting them with detergents or ultrasound, indicating that the active site of pyrophosphatase is located in the matrix. Initial rates of PPi hydrolysis by toluene-permeabilized mitochondria and purified pyrophosphatase were found to depend in a similar manner on PPi and Mg2+ concentrations. The simplest model consistent with the data in both cases implies that the reaction proceeds through two pathways and requires MgPPi as the substrate and, at least, one Mg2+ ion as the activator. In the presence of 0.4 mM Mg2+ (physiological concentration), the inhibition is, at constant for Ca2+ is 12 PM and the enzyme activity least, 50% maximal. The results suggest that the activity of pyrophosphatase in mitochondria is high enough to keep free PPi concentration at a level close to that at equilibrium. 0 1992 Academic Press, Inc.

Fatty acid oxidation in mitochondria results in concomitant production of pyrophosphate, which is an important regulator of a number of vital reactions. PPi inhibits matrix acyl-CoA synthetase (l), a key enzyme of fatty acid metabolism, and controls matrix volume, which regulates respiration, ATP production, pyruvate carboxylation, and other mitochondrial functions (2, 3). Mitochondria contain about 90% of cellular PPi (4-6), which can only slowly cross the inner mitochondrial membrane in exchange for adenine nucleotides (7, 8) and cannot participate in metabolic pathways without prior hydrolysis to Pi. The concentration of PPi in isolated mitochondria is about 0.1 mM (2, 4, 9), which is by several orders of magnitude higher than that predicted on the basis of the 2Pi G PPi equilibrium (10). This is quite surprising since mitochondria contain very active pyrophosphatase (ll-14), which, when working at its full capacity, would hydrolyze matrix PPi in less than 1 s. It seems thus that either pyrophosphatase activity is de-

creased in mitochondria or PPi is not available to the enzyme. In this work we addressed this problem by examining the catalytic properties of pyrophosphatase within mitochondria permeabilized to low molecular weight solutes. MATERIALS



Rat liver mitochondria were isolated according to Fleischer et al. (15) and suspended in 10 mM potassium 4-(2-hydroxyethyl)-l-piperazinethanesulfonate buffer (pH 7.4) containing 0.3 M sucrose. Submitochondrial particles were prepared as described by Wherle et al. (16). Solubilization of the mitochondria was performed by incubating them at a concentration of 10 mg protein/ml with 0.2% Triton X-100 or 0.4% sodium cholate for 2 min at 4°C. Permeabilization of the mitochondria with toluene was done according to Rutter and Denton (17). Mitochondrial inorganic pyrophosphatase with a specific activity of 33 IU/mg was isolated in this laboratory by Dr. A. Unguryte as described elsewhere (14). Initial rates of PP, hydrolysis were estimated from continuous recordings of P, formation obtained with an automatic analyzer (18) at a sensitivity of 3 ELM Pi per recorder scale. PP, hydrolysis was initiated by adding 5-50 ~1 of enzyme solution to a 17-ml reaction mixture containing 0.1 M Tris-HCl (pH 7.2), 25 FM ethylene glycol bis(Saminoethy1 ether)N,N’-tetraacetate, and varying amounts of MgClz and NalP,O, at 25°C. When mitochondria or submitochondrial particles were assayed, this medium was supplemented with 0.3 M sucrose. Rate data were corrected for nonenzymatic PP, hydrolysis. All runs were repeated two to four times, and the rate data obtained usually agreed within 5-10%. Enzyme concentration was varied in order to obtain nearly equal measured rates at all sets of MgPP; and Mg2+ concentrations. Procedures used to calculate total concentrations of PP, and MgC& required to maintain desired levels of MgPP, and Mg2+ were as described previously (19). Data treatment was performed using a program for nonlinear regression (20). Rate values were weighted according to l/u*. Polyacrylamide gel electrophoresis at pH 8 was carried out using a modified (13) procedure of Williams and Reisfeld (21). The gels were stained for pyrophosphatase activity (22). Protein was measured according to Lowry et al. (23) in the presence of 1% sodium deoxycholate using bovine serum albumin as the standard.

RESULTS Pyrophosphatase


Activity of Mitochondria Particles


Treatment of mitochondria with toluene stimulated their pyrophosphatase activity approximately lo-fold and

16 All

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essentially to the same deg:ree (0.09 IU/mg at 0.5 mM PPi, 2 mM MgCl,) as did sonication and treatment with Triton X-100 or sodium cholate. .About 90% of the activity was found in the sediment upon centrifugation at 10,OOOgfor 10 min, indicating that this stimulation was due to the ability of PPi to cross the membrane of the toluene-treated mitochondria rather than to release of the enzyme into solution. In contrast, pyrophosphatase activity of submitochondrial particles prepared by sonication and differential centrifugation of mitochondria (16) was the same in the absence and in the presence of Triton X-100 (0.03-0.05 IU/mg). In such particles, the former matrix side of the inner membrane is orientated into solution (16). Mitochondria which have not been subjected to any further treatment expressed 6 to 12% of the activity apparent in the mitochondrial extracts and toluene-treated mitochondria. Schick and Butler (24) suggested that this activity is due to membrane damage during the isolation procedure, and this idea is supported by the following observations. All activity expressed by isolated mitochondria was found in supernatant on centrifugation of their suspension in the isolation medium at 10,OOOgfor 10 min. The pellet which was resuspended with a Teflon pestle, however, expressed the same activity as before the centrifugation. If, on the other hand, the centrifugation was done using a 15-45% sucrose gradient to prevent mitochondria from reaching the bottom of the centrifuge tube and forming a dense pellet, their activity dropped to 2%. This means that partial damage to mitochondria with concomitant unmasking of some pyrophosphatase activity occurred during the resuspension procedure.





FIG. 1. Dependence of the rate of PP, hydrolysis by permeabilized mitochondria on magnesium pyrophosphate concentration in the presence of 0.05 (O), 0.1 (O), 0.2 (A), 0.5 (A), 2 (El), 5 (m), and 20 (0) mM free Mg’+. The lines show the best fit for the model in Scheme I.

the maximal velocities for the two pathways. The plot of which could be constructed using the l/vapp vs l/[Mg”], data given in Table I, was concave upward, indicating that the values of V and V’ are different.


Kl E-r--EMz



+EM,(MPP) $v,


Steady-State Kinetics of PPi Hydrolysis by Permeabilized Mitochondria and Purified Enzyme Initial rates of PPi hydrolysis by pyrophosphatase within toluene-treated mitochondria were measured in the presence of 0.2-100 ph/l MgPPi and 0.05-20 mM free Mg 2+. Part of the data is shown in Fig. 1. Initial rates of PPi hydrolysis by the purified pyrophosphatase were measured in the presence of l-100 /.LM MgPPi and 0.0320 mM Mg’+. In both cases, the reaction followed Michaelis-Menten kinetics at fixed levels of free Mg’+. The values of the apparent maximal velocity and of the ratio of the apparent Michaelis constant to it (Table I) depended on free Mg2+ concentration in a manner similar to that reported earlier for the purified mitochondrial pyrophosphatase at pH 8.5 (25). The rate data were analyzed in terms of the general model used previously for rat liver cytosolic pyrophosphatase and mitochondrial pyrophosphatases at pH 8.5 (25). The model, however, could be simplified as shown in Scheme I. It implies that the reaction proceeds through two pathways, the actual substrate is MgPPi and free Mg2+ is an essential activator. Kl and K2 are the dissociation constants for the enzyme metal complexes, K, and KL are the Michaelis constants, and V and V’ are



The rate equation for this model is given by (25)

The kinetic parameters were obtained by direct fitting this equation to all rate data (214 and 158 independent measurements for the permeabilized mitochondria and purified enzyme, respectively) and are given in Table II. The value of Kl for the permeabilized mitochondria was less than 0.05 mM, the lowest concentration of Mg2+ used, and for this reason could not be determined with reasonable precision. In contrast, the value of Kl for the purified pyrophosphatase was considerably higher and E was a kinetically significant species as indicated by a 2.5-fold increase in the sum of the squares of residuals at Kl = 0. This model seems to be the simplest one consistent with the data. Excluding any of the species made the fit significantly worse while adding other species did not affect it. No systematic deviation was observed, and the




TABLE Michaelis-Menten



v am

Free MS+



(IU mg-i)

20 10 5 2


for the Hydrolysis of Magnesium Pyrophosphate by Permeabilized Purified Pyrophosphatase at Different M2+ Concentrations Permeabilized





0.092 f 0.004

5.5 f 0.3 7.6 + 0.9

0.100 * 0.003

10.9 f 1.2

0.077 f 0.005

26 f 4

0.091 f 0.002


0.095 f 0.087 k 0.050 k 0.039 f 0.032 f

0.675 0.5 0.2 0.1 0.05

0.004 0.003 0.003 0.003 0.002

50 + 4 74 + 3 141f 14 260 f 30 410 +43

mean relative deviation of the predicted vs measured rate was about 12% in both cases, which compared well with the precision of rate measurements. It is seen from Table II that the binding and Michaelis constants for the mitochondria-bound and purified pyrophosphatase are quite close, except for Ki . Inhibition of Pyrophosphatase Mitochondria by Ca”

in Permeabilized

Ca2+ strongly inhibited pyrophosphatase in toluenetreated mitochondria (Fig. 2). The concentration of Ca2+ causing a 50% effect in the presence of 0.4 mM Mg2+ was about 12 pM, which is close to the values obtained for the purified enzyme (26) and mitochondrial extracts (27). DISCUSSION

Toluene treatment has been previously shown to unmask the activities of several matrix dehydrogeneses in rat heart mitochondria (17,28). When applied to rat liver mitochondria, this procedure also resulted in a stable, permeabilized preparation in which the activity of pyro-





v aw


(IU rng-i)


35.3 * 34 f 35.5 f 31fl 31f

1 1

0.024 + 0.003 0.025 + 0.003 0.052 + 0.012


0.11 f 0.005 0.174 f 0.007

22 f 20 * 20 f 18 +


mg IU’)

0.220 250.012 0.73 f 0.04


1 1

1.01 Zk 0.05


2.13 f 0.16

phosphatase, latent in intact mitochondria, was expressed to 100%. The enzyme remained within permeabilized mitochondria, as indicated by their cosedimentation. The activity of pyrophosphatase could be also unmasked by disruption of mitochondria with detergents or ultrasound. If one takes into account the fact that intact mitochondria are impermeable to PPi (l), these results strongly indicate that the active site of pyrophosphatase is located in the matrix. Rat liver mitochondria contain pyrophosphatase in both the inner membrane and the matrix (6). This could have complicated the kinetic analysis but the matrix enzyme represents only 20% of total activity. Besides, kinetic properties of the two enzymes are quite similar, as shown earlier for the bovine heart mitochondria (19), because the matrix enzyme is the catalytic part of the membrane enzyme (13). The kinetic model for the mitochondrial pyrophosphatase at pH 7.2 derived in this work is similar to but not


Summary of the Kinetic Parameters for PP, Hydrolysis by Permeabilized Mitochondria and Purified Pyrophosphatase at pH 7.2 (25°C)


Permeabilized mitochondria

Kl bM)

K2 bM) Km (PM) K:, (PM) V IU (mg-‘) V’ IU (mg-‘)

13 f 20 f 0.30 f 0.023 f 0.089 f

1.6 2 0.03 0.002 0.002

Purified pyrophosphatase

0.10 + 0.04 19 -c 5 15 rt 2 0.4 f 0.1 17.2 f 1 37.4 + 1.2




FIG. 2. Inhibition of pyrophosphatase activity in permeabilized mitochondria by Ca*+ m the presence of 0.4 mM Mg’+. PP, concentration was 10 pM.



identical with that reported previously for this enzyme at pH 8.5 (25). When analyzing the rate data obtained at pH 8.5, we postulated that V is equal to V’, in fact, assuming thereby that there is only one pathway for conversion of enzyme-bound PPi. The dependence of Vapp on Mg2+ concentration at pH 7.2 clearly indicates that the reaction can proceed through two pathways with different maximal velocities. In this respect, the mitochondrial enzyme is similar to the yeast pyrophosphatase, for which the existence of two pathways stems from both PPi hydrolysis (29) and oxygen exchange (30) data. The kinetic pattern of oxygen exchange between Pi and water by the mitochondrial pyrophosphatase is also in full harmony with the idea of two reaction pathways (Smirnova, I. N., and Kasho, V. N., unpublished). The two enzymes differ, however, in that V > V’ for the yeast one (29). The activity of the mitochondrial pyrophosphatase in the presence of 0.4 mM Mg2+ (expected physiological concentration (31)) and excess PPi can be estimated from the equation given above to be 75% maximal. The concentration of free Ca2+ in lmitochondria can hardly exceed several micromoles per liter (32-35), i.e., much lower compared to the Ki for this inhibitor. This means that pyrophosphatase can be, at least, 50% as active in mitochondria, which corresponds to the rate of PPi hydrolysis of 45 nmol/min per milligram protein. It has been reported that isolated mitochondria contain approximately 0.1 nmol lDPi per milligram protein (2, 4, 7), which is in accord w:ith our own estimates (unpublished). This PPi level is not changed markedly when mitochondria are kept at 37°C for at least 10 min, although one can easily calculate that the activity of the enzyme is enough to destroy all .PPi in less than 1 s. The most likely explanation for this apparent inconsistency is that PPi may not be available to the enzyme. It is supported by the observation that no PPi-attributable signal was observed in 31P NMR st.udies of rat liver although the PP; concentration in mitochondria was sufficient to be detected if it were free in solution (36). The mechanism of PPi sequestration in mitochondria can only be guessed at. Binding to calcium phLosphate granules, proteins, and membranes is the most likely alternative. One cannot exclude, however, that PPi hydrolysis may be inhibited by some mitochondrial constituent or by a membrane potential whose changes are supposedly coupled to PPi hydrolysis (37). The latter alternative seems, however, less likely because the matrix pyrophosphatase, which accounts for approximately 20% of total mitochondrial activity, cannot be affected in this way. REFERENCES 1. Batenburg, J. J., and van den Bergh, S. G. (1972) Biochim. Biopkys. Acta 280,495-505. 2. Davidson, A. M., and Halestrap, A. P. (1987) Biochem. J. 246,715723. 3. Halestrap, A. P. (1989) Biochim. Biophys. Acta 973, 355-382.



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Catalytic properties of the inorganic pyrophosphatase in rat liver mitochondria.

Intact rat liver mitochondria have very low hydrolytic activity, if any, toward exogenous pyrophosphate. The activity can be unmasked by making mitoch...
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